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Single-particle analysis using cryo-electron microscopy has emerged recently as a tool for elucidating the structure of biological macromolecules and their assemblies. A prerequisite for single-particle analysis is an ensemble of images of structurally identical particles in different orientations. There are a variety of techniques used for image processing of this type of object in electron microscopy. The paper gives an overview of the general philosophy of image analysis of single particles in electron microscopy. It has been shown that multivariate statistical analysis of large data sets in conjunction with angular reconstitution is capable of yielding a structural resolution approaching that of X-ray structural analysis of large macromolecules. Structure preservation during specimen preparation for electron microscopy is crucial for high-resolution studies. Nowadays, cryo-electron microscopy is a beneficial method for studying biological macromolecules in their natural environment, allowing their rapid freezing in a particular functional state. The processing of images of 50S Escherichia coli ribosomal subunits embedded in vitreous ice is used as an example of image analysis of single particles at 7.5 A resolution.

In this paper the question was studied whether Escherichia coli ribosomes which have undergone a specific cleavage of their 16-S rRNA by treatment with the bacteriocin cloacin DF13, are able to enter and complete an elongation cycle. As we have shown in the preceding paper in this journal, these defective ribosomes can form 70-S initiation complexes with MS2 RNA as a messenger. Binding of the second aminoacyl-tRNA (alanyl-tRNA) to the latter complexes led to the formation of fMet-Ala-tRNA which was fully puromyein-sensitive. In addition to these aminoacyl-tRNAs also the third aminoacyl-tRNA (seryl-tRNA) can be bound to defective ribosomes. Dual-label experiments showed that puromycin released all radioactivity from the ribosomes. This indicates that fMet-Ala-Ser-tRNA is formed and is translocated from the A site to the P site. Control ribosomes bind the three aminoacyl-tRNAs in a 1:1:1 ratio. The defective particles bind fMet-tRNA, Ala-tRNA and Ser-tRNA in a ratio of 1:0.5:0.25. Kinetic experiments strongly suggest that at each binding of the ternary complex aminoacyt-tRNA · EF-Tu · GTP to the A site of defective ribosomes, about half of these particles undergo irreversible inactivation. Consequently polypeptide synthesis under our conditions will come to an end after incorporation of only four or five amino acids. The particles which temporarily survive ternary complex binding are fully capable of transpeptidation and translocation. [ABSTRACT FROM AUTHOR]

Summary: Different codon optimization algorithms are available that aim at improving protein production by optimizing translation elongation. In these algorithms, it is generally not considered how the altered protein coding sequence will affect the secondary structure of the corresponding RNA transcript, particularly not the effect on the 5′‐UTR structure and related ribosome binding site availability. This is a serious drawback, because the influence of codon usage on mRNA secondary structures, especially near the start of a gene, may strongly influence translation initiation. In this study, we aim to reduce the effect of codon usage on translation initiation by applying a bicistronic design (BCD) element. Protein production of several codon‐optimized gene variants is tested in parallel for a BCD and a standard monocistronic design (MCD). We demonstrate that these distinct architectures can drastically change the relative performance of different codon optimization algorithms. We conclude that a BCD is indispensable in future studies that aim to reveal the impact of codon optimization and codon usage correlations. Furthermore, irrespective of the algorithm used, using a BCD does improve protein production compared with an MCD. The overall highest expression from BCDs for both GFP and RFP is at least twofold higher than the highest levels found for the MCDs, while for codon variants having very low expression from the MCD, even 10‐fold to 100‐fold increases in expression were achieved by the BCD. This shows the great potential of the BCD element for recombinant protein production. This paper outlines the improved protein expression when using a bicistronic design (BCD) in Escherichia coli. Additionally, the decoupling of translation initiation from translation elongation by a BCD changes the relative performance of different codon optimization algorithms. [ABSTRACT FROM AUTHOR]

UTP-modulated attenuation of transcription is involved in regulating the synthesis of pyrimidine nucleotides in Escherichia coli. Thus, expression of two genes, pyrBI and pyrE, was shown to be under this type of control. The genes encode the two subunits of aspartate transcarbamylase and orotate phosphoribosyltransferase respectively. The levels of these enzymes are inversely correlated with the intracellular concentration of UTP. Modulation of attenuation seems to be a consequence of the effect of UTP concentration on the mRNA chain growth rate. Reducing the UTP pool retards RNA polymerase movement. Mechanistically this will couple the ribosomes translating a leader peptide gene more tightly to the elongating RNA polymerase. The ribosomes will then be more prone to prevent the folding of the mRNA chains into terminating hairpin structures when RNA polymerase is at the attenuator and has to decide whether transcription should terminate or continue into the structural genes. This paper described a study of pyrBI and pyrE gene regulation in cells where the ribosomes move slowly as a result of mutation in rpsL. It appears that expression of the two genes is hyper-regulated by the UTP pool in this type of cells. Furthermore, the attenuator model can only account for the results if it is assumed that UTP-concentration dependent pausing of transcription occurs in vivo in the two pry gene leaders such that RNA polymerase waits for the coupled ribosomes before transcribing into the attenuator regions. [ABSTRACT FROM AUTHOR]

We have incorporated chloroplast ribosomal proteins (R-proteins) L12 and S18 into Escherichia coli ribosomes and examined the hybrid ribosomes for their ability to form polysomes in vivo and perform poly(U)-dependent poly(Phe) synthesis in vitro. The rye chloroplast S18 used for the experiment is a highly divergent protein (170 amino acid residues; E. coli S18, 74 residues), containing a repeating, chloroplast-specific, heptapeptide motif, and has amino acid sequence identity of only 35% to E. coli S18. When expressed in E. coli, chloroplast S18 was assembled in E. coli ribosomes. The latter formed polysomes in vivo at about the same rate as the host ribosomes, indicating that the replacement of E. coli S18 with its chloroplast homologue has only a minor, if any, effect on function. The L12 protein is much more conserved in sequence and chain length, and is known to have a very important function. The Arabidopsis chloroplast L12 used in the experiment was incorporated into E. coli 50S subunits that associated with the 30S subunits to form ribosomes, but the latter were unable to form polysomes. This result indicates functional inactivation of E. coli ribosomes by a chloroplast R-protein. To further confirm this result, we overproduced chloroplast L12 through the use of a secretion, vector and purified the protein to homogeneity. Chloroplast L12 could be efficiently incorporated in vitro into L7/12-1acking E. coli ribosomes, but the hybrid ribosomes were totally inactive in poly(U)-dependent poly(Phe) synthesis. Computer modeling of the spatial structure of all known chloroplast L12 proteins (using E. coli L12 coordinates) indicated a 'chloroplast loop' present only in chloroplast L12. The presence of this loop might have a role in the observed inactivation. Taken together with previously reported results (summarized in this paper), it would appear that the features of chloroplast R-proteins concerned with specific functions are more divergent than their assembly properties. We have previously described methods suitable for overproduction and purification of chloroplast Rproteins that are encoded in organellar DNA (≈20), but that gave poor yield for those encoded in the nuclear DNA (≈45). Here we describe a method that overcomes this problem and allows the purification of nucleus-encoded chloroplast R-proteins in milligram quantities. [ABSTRACT FROM AUTHOR]

We show in the present paper that the cleavages initiating decay of the ompA mRNA are suppressed both in the Escherichia coli amsts strain (originally defined by a prolonged bulk mRNA half-life) and in the rnets strain (originally defined by aberrant 9S RNA processing). The temperature-sensitive defects of both these strains are complemented by a recombinant lambda phage containing a genomic segment that carries the putative ams locus. A 5.8kb fragment from this genomic DNA segment was cloned into a low-copy plasmid and used to transform the amsts and rnets strains. This resulted in growth at the non-permissive temperature and a reoccurrence of the cleavages initiating decay of the ompA mRNA. Deletion analyses of this 5.8 kb fragment indicated that the putative ams open reading frame could complement both the Amsts and the Rnets phenotype with regard to the ompA cleavages. In addition we showed that the amsts strain suppresses 9S RNA processing to 5S RNA to the same extent as the rnets strain, and that the rnets strain has a prolonged bulk mRNA half-life, as was reported for the amsts strain. Therefore we suggest that ams and rne reflect the same gene locus; one which is involved both in mRNA decay and RNA processing. We discuss how this gene locus may related to the previously characterized endoribonucleolytic activities of RNase E and RNase K. [ABSTRACT FROM AUTHOR]

The complete nucleotide sequence of the 16-S RNA from Escherichia coli has been determined using rapid RNA-sequencing gel methods. The experimental data are fully described in this paper. The specificities of the ribonucleases, especially the ribonuclease PhyI are discussed and the consequences of the persistence of stable secondary structure are considered. The proposed sequence contains 1541 nucleotides and agrees completely with the DNA sequence of the rrnB cistron deduced by Brosius, J., Palmer, M. L., Kennedy, P.J., and Noller, H. F. [Proc. Natl Acad. Sci. U.S.A. (1978) 75, 4801–4805]. But there are several cistron heterogeneities of which we described 16 single-base heterogeneities, 7 of the deletion/insertion type and 9 of the transition or transversion type. Our observations suggest the existence, among the 7 ribosome RNA cistrons, of one or two mutated ones. The respective advantages and disadvantages of both RNA and DNA sequencing methods are discussed. [ABSTRACT FROM AUTHOR]

Virginiamycin S is an inhibitor of protein synthesis in vivo. In this paper we show by equilibrium dialysis that it binds specifically to the 50-S subunit of Escherichia coli ribosomes, with one binding site per subunit. This binding is not altered by the presence of chloramphenicol, tetracycline or puromycin but is competed for by erythromycin. Using the splitting-reconstitution method, it could be demonstrated that protein L16 is absolutely required for the binding of virginiamycin S to the 50-S subunit. [ABSTRACT FROM AUTHOR]

The puromycin analogue. 5'-O-(N-bromoacetyl-p-aminophenylphosphoryl)-3'-N-L-phenylalanyl puromycin aminonucleoside, acts as an affinity label in covalently binding to 23-S RNA in the active centre of peptidyl transferase of Escherichia coli ribosomes [Greenwell, P., Harris, R. J. & Symons. R. H. (1974) Eur. J. Biochem. 49, 539-554]. The sequence of a pentanucleotide fragment of 23-S RNA to which the affinity label is attached has now been determined. In an attempt to determine the nucleotide residue to which the affinity label was attached, a 32Plabelled affinity label was reacted with ribosomal RNA, poly(A), poly(C), poly(G) and poly(U) under the conditions used for affinity labelling of ribosomes. However, no specific reaction occurred, confirming our earlier observation that this required the functional integrity of the ribosome. An alternative approach of affinity labelling ribosomes labelled by incubating E. coli in the presence of [³H]adenosine, [³H]cytidine, [³H]guanosine or [³H]uridine, showed that the affinity label was attached to a CMP residue. Effinity-labelled oligonucleotide fragments were purified for sequence analysis as follows. The free α-amino group of the affinity-label molecule attached to 23-S ribosomal [32P]RNA was coupled to biotin by reaction with the N-hydroxysuccinimide ester of biotin. After digestion of the RNA with either ribonuclease A or T1, the basic protein avidin was added to form the avidin, biotin-affinity-label-oligonucleotide fragment. Stepwise chromatography on phosphocellulose, followed (after dissociation of the avidin) by electrophoresis on DEAE-cellulose at pH 1.9 gave substantial purification of the biotin-affinity-label-oligonucleotide. From the nucleotide composition of the purified ribonuclease A and T1 fragments, the pentanucleotide sequence of G-U-C*-C-Gp was obtained, where C* represents C modified by the affinity label. [ABSTRACT FROM AUTHOR]

When Escherichia coli 30-S ribosomal subunits are hydrolysed under mild conditions, two ribonucleoprotein fragments of unequal size are produced. Knowledge of the RNA sequences contained in these hydrolysis products was required for the experiments described in the preceding paper, and the RNA sub-fragments have therefore been examined by oligonucleotide analysis. Two well-defined small fragments of free RNA, produced concomitantly with the ribonucleoprotein fragments, were also analysed. The larger ribonucleoprotein fragment, containing predominantly proteins S4, S5, S8, S15, S16 (17) and S20, contains a complex mixture of RNA sub-fragments varying from about 100 to 800 nucleotides in length. All these fragments arose from the 5′-terminal 900 nucleotides of 16-S RNA, corresponding to the well-known 12-S fragment. No long-range interactions could be detected within this RNA region in these experiments. The RNA from the smaller ribonucleoprotein fragment (containing proteins S7, S9, S10, S14 and S19) has been described in detail previously, and consists of about 450 nucleotides near the 3′ end of the 16-S RNA, but lacking the 3′-terminal 150 nucleotides. The two small free RNA fragments (above) partly account for these missing 150 nucleotides; both fragments arose from section A of the 16-S RNA, but section J (the 3′-terminal 50 nucleotides) was not found. This result suggests that the 3′ region of 16-S RNA is not involved in stable interactions with protein. [ABSTRACT FROM AUTHOR]

The crystal structure of Thermus thermophilus ribosomal protein TL5 in complex with a fragment of Escherichia coli 5S rRNA has been determined at 2.3 Å resolution. The protein consists of two domains. The structure of the N-terminal domain is close to the structure of E. coli ribosomal protein L25, but the C-terminal domain represents a new fold composed of seven β-strands connected by long loops. TLS binds to the RNA through its N-terminal domain, whereas the C-terminal domain is not included in this interaction. Cd2+ ions, the presence of which improved the crystal quality significantly, bind only to the protein component of the complex and stabilize the protein molecule itself and the interactions between the two molecules in the asymmetric unit of the crystal. The TLS sequence reveals homology to the so- called general stress protein CTC. The hydrophobic cores which stabilize both TLS domains are highly conserved in CTC proteins. Thus, all CTC proteins may fold with a topology close to that of TL5. [ABSTRACT FROM AUTHOR]

A resistance gene, carB, originally isolated from the carbomycin-producing organism. Streptomyces thermotolerans, confers on Streptomyces lividans high-level resistance to the drug. However, ribosomes from S. lividans expressing carB show only moderate resistance to this macrolide in vitro, although they are highly resistant to the action of lincosamide antibiotics. The carB product monomethylates the amino group of the adenosine residue located at position 2058 in 23S ribosomal RNA. In contrast, ribosomes from S. lividans expressing ermE, in which 23S RNA is dimethylated at this same position, are much more highly resistant to macrolides and insensitive to lincosamides. [ABSTRACT FROM AUTHOR]

A ribonucleoprotein prepared by strong ribonuclease digestion of a complex of 16-S ribosomal RNA and proteins S4 and S20 from Escherichia coli has been characterized; its nucleotide sequence, the positions of enzyme cuts and the sequence excisions have been placed in the completed sequence of 16-S RNA. The positions and yields of enzyme cuts, and excisions of sequence, are compared with those of various ribonucleoproteins prepared with S4 or S20 alone, and with the ribonuclease-resistant S4 RNA prepared from renatured 16-S RNA in the absence of ribosomal protein. These data yield important information on the topography and organization of the 5′ third of the 16-S RNA which is selectively maintained in its native conformation by the bound proteins; they also provide criteria for testing secondary structural models of this region of 16-S RNA. [ABSTRACT FROM AUTHOR]

The effect of bacteriocin (cloacin DF13) treatment of Escherichia coli ribosomes on initiation of protein synthesis has been studied in detail. In agreement with out previous findings [Baan et al. (1976) Proc. Natl Acad. Sci. U.S.A, 73, 702–706] it is shown that 70-S initiation complexes can be formed with cloacin-treated ribosomes, but that the initiation factor IF-1 does not function properly. The following pleiotropic effects of this factor have been studied: (a) the acceleration of ribosomal subunit exchange with 70-S couples; (b) the stimulation of the IF-3-mediated dissociation of 70-S ribosomes; (c) the stimulation of 30-S initiation complex formation; (d) the enhancement of the rate of release of IF-2 from 70-S initiation complexes. The effects (a) and (b) are virtually abolished after cleavage of 16-S rRNA. The effect (d) is only partially reduced whereas effect (c) seems to be unimpaired. It is concluded that 70-S initiation complex formation with cloacin-treated ribosomes suffers from improper functioning of IF-1 in the generation of active subunits from 70-S tight couples. This is the only effect on initiation. It can he compensated for by adding more IF-3. The data provide functional evidence that 16-S rRNA is involved in ribosomal subnnit interaction. [ABSTRACT FROM AUTHOR]

After digestion of protein S4 with trypsin, all 32 tryptic peptides were isolated. Their amino acid compositions were analyzed and the sequence of the amino acids within the tryptic peptides was determined by means of a solid-phase peptide sequenator and by exopeptidases. Alignment of the tryptic peptides was established by analyzing and partially sequencing peptides isolated after digestion of the $4 protein with chymotrypsin, thermolysin and a glutamic-acid-specific protease. Further information about the alignment of peptides came from treatment of S4 with CNBr and with a lysine-modifying reagent. Protein S4 consists of 203 amino acids (Asp8, Asn8, Thr6, Ser10, Glu16, Gln10, Pros, Gly15, Ala18, Va116, Cys1, Met3, Ile8, Leu20, Tyr8, Phe4, His3, Lys20, Arg22 and Trp1) and has a molecular weight of 22550. The basic amino acids are clustered in five regions. Many short repetitions mainly with charged amino acids occur. A prediction is made for regions with or α-helices and with β-sheets. [ABSTRACT FROM AUTHOR]

Canonically, tRNA synthetases charge tRNA. However, the lysyl‐tRNA synthetase paralog EpmA catalyzes the attachment of (R)‐β‐lysine to the ε‐amino group of lysine 34 of the translation elongation factor P (EF‐P) in Escherichia coli. This modification is essential for EF‐P‐mediated translational rescue of ribosomes stalled at consecutive prolines. In this study, we determined the kinetics of EpmA and its variant EpmA_A298G to catalyze the post‐translational modification of K34 in EF‐P with eight noncanonical substrates. In addition, acetylated EF‐P was generated using an amber suppression system. The impact of these synthetically modified EF‐P variants on in vitro translation of a polyproline‐containing NanoLuc luciferase reporter was analyzed. Our results show that natural (R)‐β‐lysylation was more effective in rescuing stalled ribosomes than any other synthetic modification tested. Thus, our work not only provides new biochemical insights into the function of EF‐P, but also opens a new route to post‐translationally modify proteins using EpmA. [ABSTRACT FROM AUTHOR]

Ribosome recycling factor (RRF) plays a central role during the recycling of ribosomes in the final step of protein biosynthesis in prokaryotes and is therefore a favourable target for the development of new antibiotics. The crystal structure of Escherichia coli RRF has been reported to have an open L-shaped conformation, while other RRFs from thermophilic bacteria have a strict L-shaped conformation [Yun et al. (2000), Acta Cryst. D56, 84-85]. Wild-type E. coli RRF has so far not been crystallized free from bound detergent. Here, a mutant of RRF, Arg132Gly, has been crystallized without any detergent. A complete data set from a crystal of this mutant obtained by the hanging-drop vapour-diffusion method has been collected at 2.2 Å resolution using synchrotron radiation at 100 K. The crystal belongs to the monoclinic space group P21, with unit-cell parameters a = 46.02, b = 49.27, c = 49.37 Å, β = 110.1°. The currently refined structure indicates that RRF has a tRNA-like L-shaped conformation. [ABSTRACT FROM AUTHOR]

Summary: Transcription and translation are coupled processes in bacteria. A role of transcription elongation cofactor NusG in coupling has been suggested by in vitro structural studies. NMR revealed association of the NusG carboxy‐terminal domain with S10 (NusE), implying a direct role for NusG as a bridge linking RNAP and the lead ribosome. Here we present the first in vitro and in vivo evidence of full‐length NusG association with mature 70S ribosomes. Binding did not require accessory factors in vitro. Mutating the NusG:S10 binding interface at NusG F165 or NusE M88 and D97 residues weakened NusG:S10 association in vivo and completely abolished it in vitro, supporting the specificity of this interaction. Mutations in the binding interface increased sensitivity to chloramphenicol. This phenotype was suppressed by rpoB*35, an RNAP mutation that reduces replisome‐RNAP clashes. We propose that weakened NusG:S10 interaction leads to uncoupling when translation is inhibited, with resulting RNAP backtracking, replication blocks and formation of lethal DNA double‐strand breaks. [ABSTRACT FROM AUTHOR]

Macrolides, one of the most prescribed classes of antibiotics, bind in the bacterial ribosome's polypeptide exit tunnel and inhibit translation. However, mutations and other ribosomal modifications, especially to the base A2058 of the 23S rRNA, have led to a growing resistance problem. Here, we have used molecular dynamics simulations to study the macrolides erythromycin and azithromycin in wild-type, A2058G-mutated, and singly or doubly A2058-methylated Escherichia coli ribosomes. We find that the ribosomal modifications result in less favorable interactions between the base 2058 and the desosamine sugar of the macrolides, as well as greater displacement of the macrolides from their crystal structure position, illuminating the causes of resistance. We have also examined four azithromycin derivatives containing aromatic indole-analog moieties, which were previously designed based on simulations of the stalling peptide SecM in the ribosome. Surprisingly, we found that the studied moieties could adopt very different geometries when interacting with a key base in the tunnel, A751, possibly explaining their distinct activities. Based on our simulations, we propose modifications to the indole-analog moieties that should increase their interactions with A751 and, consequently, enhance the potency of future azithromycin derivatives. [ABSTRACT FROM AUTHOR]

Bacterial growth environment strongly influences the efficacy of antibiotic treatment, with slow growth often being associated with decreased susceptibility. Yet in many cases, the connection between antibiotic susceptibility and pathogen physiology remains unclear. We show that for ribosome-targeting antibiotics acting on Escherichia coli, a complex interplay exists between physiology and antibiotic action; for some antibiotics within this class, faster growth indeed increases susceptibility, but for other antibiotics, the opposite is true. Remarkably, these observations can be explained by a simple mathematical model that combines drug transport and binding with physiological constraints. Our model reveals that growth-dependent susceptibility is controlled by a single parameter characterizing the 'reversibility' of ribosome-targeting antibiotic transport and binding. This parameter provides a spectrum classification of antibiotic growth-dependent efficacy that appears to correspond at its extremes to existing binary classification schemes. In these limits, the model predicts universal, parameter-free limiting forms for growth inhibition curves. The model also leads to non-trivial predictions for the drug susceptibility of a translation mutant strain of E. coli, which we verify experimentally. Drug action and bacterial metabolism are mechanistically complex; nevertheless, this study illustrates how coarse-grained models can be used to integrate pathogen physiology into drug design and treatment strategies. [ABSTRACT FROM AUTHOR]

Bacteria must constantly adapt their growth to changes in nutrient availability; yet despite large-scale changes in protein expression associated with sensing, adaptation, and processing different environmental nutrients, simple growth laws connect the ribosome abundance and the growth rate. Here, we investigate the origin of these growth laws by analyzing the features of ribosomal regulation that coordinate proteome-wide expression changes with cell growth in a variety of nutrient conditions in the model organism Escherichia coli. We identify supply-driven feedforward activation of ribosomal protein synthesis as the key regulatory motif maximizing amino acid flux, and autonomously guiding a cell to achieve optimal growth in different environments. The growth laws emerge naturally from the robust regulatory strategy underlying growth rate control, irrespective of the details of the molecular implementation. The study highlights the interplay between phenomenological modeling and molecular mechanisms in uncovering fundamental operating constraints, with implications for endogenous and synthetic design of microorganisms. [ABSTRACT FROM AUTHOR]

Csr is a conserved global regulatory system that controls expression of several hundred Escherichia coli genes. CsrA protein represses translation of numerous genes by binding to mRNA and inhibiting ribosome access. CsrA also activates gene expression, although an activation mechanism has not been reported. CsrA activates flhDC expression, encoding the master regulator of flagellum biosynthesis and chemotaxis, by stabilizing the m RNA. Computer modelling, gel mobility shift and footprint analyses identified two CsrA binding sites extending from positions 1-12 ( BS1) and 44-55 ( BS2) of the 198 nt flhDC leader transcript. flhD ′-′ lacZ expression was reduced by mutations in csrA and/or the CsrA binding sites. The position of BS1 suggested that bound CsrA might inhibit 5′ end-dependent RNase E cleavage of flhDC m RNA. Consistent with this hypothesis, CsrA protected flhDC leader RNA from RNase E cleavage in vitro and protection depended on BS1 and BS2. Primer extension studies identified flhDC decay intermediates in vivo that correspond to in vitro RNase E cleavage sites. Deletion of these RNase E cleavage sites resulted in increased flhD ′-′ lacZ expression. Data from m RNA decay studies and quantitative primer extension assays support a model in which bound CsrA activates flhDC expression by inhibiting the 5′ end-dependent RNase E cleavage pathway. [ABSTRACT FROM AUTHOR]

The tmRNA/ SmpB system, which is almost universal in bacteria, rescues bacterial ribosomes stalled at the end of non-stop mRNAs ( mRNAs lacking a stop codon). In addition, a few bacteria, including Escherichia coli, have developed a second two-component system as reported by Chadani et al. (). A small protein, ArfA of 55 amino acids (formerly called YdhL), mediates binding of release factor 2 to the ribosomal A site lacking a complete mRNA codon and thereby triggers translational termination and rescue of the stalled ribosome. [ABSTRACT FROM AUTHOR]

Ribosomal protein P0 forms a pentameric complex with two heterodimers of the flexible stalk proteins P1•P2 and plays a role in the functional interaction of eukaryotic ribosomes with translational factors. To investigate the functionality of domains of P0 characteristic to eukaryotes, we constructed various chimeras between silkworm P0 and Escherichia coli counterpart L10. Replacement of the C-terminal region of L10 with that of P0 allowed the binding of two P1•P2 heterodimers, which supported ribosomal activity dependent on eukaryotic elongation factors eEF-2/eEF-1α, but not activity dependent on bacterial factors EF-G/EF-Tu. Conversely, replacement of the C-terminal region of P0 with that of L10 allowed binding of two bacterial L12 homodimers, which resulted in a low level of activity dependent on bacterial factors. Insertion of the extended region of P0 that is absent in the bacterial counterpart into L10 did not affect L12 binding or bacterial factor-dependent activity, but deletion of this region from P0 resulted in a 40% reduction in eukaryotic factor-dependent activity. The results indicate that the C-terminal regions of P0 and L10 are responsible for binding of the cognate stalk dimers and ribosomal specificity for translation factors and suggest that the extended region participates in accessibility only for eukaryotic factors. [ABSTRACT FROM AUTHOR]

RsgA is a 30S ribosomal subunit-binding GTPase with an unknown function, shortage of which impairs maturation of the 30S subunit. We identified multiple gain-of-function mutants of Escherichia coli rbfA, the gene for a ribosome-binding factor, that suppress defects in growth and maturation of the 30S subunit of an rsgA-null strain. These mutations promote spontaneous release of RbfA from the 30S subunit, indicating that cellular disorders upon depletion of RsgA are due to prolonged retention of RbfA on the 30S subunit. We also found that RsgA enhances release of RbfA from the mature 30S subunit in a GTP-dependent manner but not from a precursor form of the 30S subunit. These findings indicate that the function of RsgA is to release RbfA from the 30S subunit during a late stage of ribosome biosynthesis. This is the first example of the action of a GTPase on the bacterial ribosome assembly described at the molecular level. [ABSTRACT FROM AUTHOR]

We have trapped elongation factor G (EF-G) from Escherichia coli in six, functionally defined states, representing intermediates in its unidirectional catalytic cycle, which couples GTP hydrolysis to tRNA–mRNA translocation in the ribosome. By probing EF-G with trypsin in each state, we identified a substantial conformational change involving its conserved switch I (sw1) element, which contacts the GTP substrate. By attaching FeBABE (a hydroxyl radical generating probe) to sw1, we could monitor sw1 movement (by ∼20 Å), relative to the 70S ribosome, during the EF-G cycle. In free EF-G, sw1 is disordered, particularly in GDP-bound and nucleotide-free states. On EF-G•GTP binding to the ribosome, sw1 becomes structured and tucked inside the ribosome, thereby locking GTP onto EF-G. After hydrolysis and translocation, sw1 flips out from the ribosome, greatly accelerating release of GDP and EF-G from the ribosome. Collectively, our results support a central role of sw1 in driving the EF-G cycle during protein synthesis. [ABSTRACT FROM AUTHOR]

In Escherichia coli strains carrying null mutations in either the dnaK or dnaJ genes, the late stages of 30S and 50S ribosomal subunit biogenesis are slowed down in a temperature-dependent manner. At high temperature (44°C), 32S and 45S particles (precursors to 50S subunits) and 21S particles (precursors to 30S subunits) accumulate. The latter are shown by 3′5′ rapid amplification of cDNA ends analysis to contain unprocessed or partially processed 16S ribosomal RNA at the 5′ end, but the 3′ end was never processed. This implies that maturation of 16S ribosomal RNA starts at the 5′-terminus, and that the 3′-terminus is only trimmed at a later step. At normal temperatures (30°C−37°C), ribosome assembly in both mutants is not arrested but is significantly delayed, as shown by pulse-chase analysis. Assembly defects are partially compensated by an overexpression of other heat-shock proteins, which occurs in the absence of their negative regulator DnaK, or by a plasmid-driven overexpression of GroES/GroEL, suggesting the involvement of a network of chaperones in ribosome biogenesis. [ABSTRACT FROM AUTHOR]

During the stationary phase of Escherichia coli growth, ribosomal structure changes drastically. Proteins RMF, YhbH, YfiA and SRA are expressed and bind to ribosome particles. In a process named ‘ribosomal hibernation,’ RMF binding induces the dimerization and subsequent inactivation of 70S ribosomes. Here, we examined the functions of YhbH and YfiA in the formation of 70S dimers using deletion mutants of YhbH and YfiA. The yfiA deletion mutant expressed YhbH and RMF in the stationary phase and formed a greater number of 100S particles than the wild-type, showing that YhbH promotes and stabilizes 100S formation. In contrast, the yhbH deletion mutant expressed YfiA and RMF and produced no 70S dimers, suggesting that YfiA prevents 70S dimer formation. Thus, YhbH and YfiA have opposite functions in 70S dimer formation. YhbH and YfiA share 40% sequence homology, suggesting that their binding sites overlap and they compete for a region proximal to the P- and A-sites on 30S subunits. In the yhbH and yfiA double deletion mutant, which expresses only RMF, 70S dimers were observed as 90S particles. Since 100S particles were seen in the yfiA deletion mutant containing RMF and YhbH, YhbH probably converts immature 90S ribosomes into mature 100S particles. [ABSTRACT FROM AUTHOR]

We have addressed the mechanism of insertion of both transmembrane segments (TMs) of leader peptidase, a double-spanning protein, into the Escherichia coli inner membrane. Using photo-crosslinking, the first TM (H1) was shown to insert at a Sec-translocon/YidC interface in a fixed orientation. H1 lost its contacts with the Sec-translocon and gained access to lipids near YidC soon after complete exposure outside the ribosome. Following lipid integration, it moved away from the Sec/YidC insertion site. The second TM (H2) inserted and interacted with SecY and YidC in a similar transient fashion. The data are consistent with a linear integration model in which the TMs of polytopic inner membrane proteins move one by one from a Sec/YidC insertion site into the lipid bilayer. We propose that YidC assists the lipid partitioning of single TMs. [ABSTRACT FROM AUTHOR]

Ribosomes in Escherichia coli change their composition and conformation in the stationary phase. Ribosome modulation factor (RMF) and ribosomal protein S22 are known to be associated with stationary phase ribosomes. RMF association causes the loss of translational activity and the dimerization of 70S ribosomes into 100S ribosomes, which may increase cell survival in the stationary phase. Two weakly acidic proteins having related amino acid sequences were found to be associated with E. coli ribosomes in the stationary phase. These proteins are the products of ORFs named yfiA and yhbH. The sum of the copy numbers of their product proteins, YfiA and YhbH, in the ribosomal particles was low in the log phase, but increased to nearly one in the stationary phase. YfiA was found in the 70S ribosomal fraction rather than the 100S. On the other hand, YhbH was detected exclusively in the 100S ribosomal fraction. When the stationary phase cells were transferred to fresh medium, YfiA and YhbH were found in the 70S ribosomal fraction, but not in the polysome fraction. Two proteins, YfiA and YhbH, associated with E. coli ribosomes were found to accumulate in the stationary phase, leading to the formation of several types of ribosomes. They are not likely to have roles in the elongation step of the translation in log phase cells, but are likely to be involved in the stabilization and preservation of ribosomes in the stationary phase, which might be necessary for cell survival. [ABSTRACT FROM AUTHOR]

Ketolides represent a new generation of macrolide antibiotics. In order to identify the ketolide-binding site on the ribosome, a library of Escherichia coli clones, transformed with a plasmid carrying randomly mutagenized rRNA operon, was screened for mutants exhibiting resistance to the ketolide HMR3647. Sequencing of the plasmid isolated from one of the resistant clones and fragment exchange demonstrated that a single U754A mutation in hairpin 35 of domain II of the E. coli 23S rRNA was sufficient to confer resistance to low concentrations of the ketolide. The same mutation also conferred erythromycin resistance. Both the ketolide and erythromycin protected A2058 and A2059 in domain V of 23S rRNA from modification with dimethyl sulphate, whereas, in domain II, the ketolide protected, while erythromycin enhanced, modification of A752 in the loop of the hairpin 35. Thus, mutational and footprinting results strongly suggest that the hairpin 35 constitutes part of the macrolide binding site on the ribosome. Strong interaction of ketolides with the hairpin 35 in 23S rRNA may account for the high activity of ketolides against erythromycin-resistant strains containing rRNA methylated at A2058. The existence of macrolide resistance mutations in the central loop of domain V and in hairpin 35 in domain II together with antibiotic footprinting data suggest that these rRNA segments may be in close proximity in the ribosome and that hairpin 35 may be a constituent part of the ribosomal peptidyl transferase centre. [ABSTRACT FROM AUTHOR]

In Escherichia coli, rRNA operons are transcribed as 30S precursor molecules that must be extensively processed to generate mature 16S, 23S and 5S rRNA. While it is known that RNase III cleaves the primary transcript to separate the individual rRNAs, there is little information about the secondary processing reactions needed to form their mature 3′ and 5′ termini. We have now found that inactivation of the endoribonuclease RNase E slows down in vivo maturation of 16S RNA from the 17S RNase III cleavage product. Moreover, in the absence of CafA protein, a homolog of RNase E, formation of 16S RNA also slows down, but in this case a 16.3S intermediate accumulates. When both RNase E and CafA are inactivated, 5′ maturation of 16S rRNA is completely blocked. In contrast, 3′ maturation is essentially unaffected. The 5′ unprocessed precursor that accumulates in the double mutant can be assembled into 30S and 70S ribosomes. Precursors also can be processed in vitro by RNase E and CafA. These data indicate that both RNase E and CafA protein are required for a two step, sequential maturation of the 5′ end of 16S rRNA, and that CafA protein is a new ribonuclease. We propose that it be renamed RNase G. [ABSTRACT FROM AUTHOR]

The structure of the Escherichia coli ribosomal protein L25 has been determined to an r.m.s. displacement of backbone heavy atoms of 0.62 ± 0.14 Å by multidimensional heteronuclear NMR spectroscopy on protein samples uniformly labeled with 15N or 15N/13C. L25 shows a new topology for RNA-binding proteins consisting of a six-stranded β-barrel and two α-helices. A putative RNA-binding surface for L25 has been obtained by comparison of backbone 15N chemical shifts for L25 with and without a bound cognate RNA containing the eubacterial E-loop that is the site for binding of L25 to 5S ribosomal RNA. Sequence comparisons with related proteins, including the general stress protein, CTC, show that the residues involved in RNA binding are highly conserved, thereby providing further confirmation of the binding surface. Tertiary structure comparisons indicate that the six-stranded β-barrels of L25 and of the tRNA anticodon-binding domain of glutaminyl-tRNA synthetase are similar. [ABSTRACT FROM AUTHOR]

Ribosome recycling factor (RRF) catalyzes the fourth step of protein synthesis in vitro: disassembly of the post-termination complex of ribosomes, mRNA and tRNA. We now report the first in vivo evidence of RRF function using 12 temperature-sensitive Escherichia coli mutants which we isolated in this study. At nonpermissive temperatures, most of the ribosomes remain on mRNA, scan downstream from the termination codon, and re-initiate translation at various sites in all frames without the presence of an initiation codon. Reinitiation does not occur upstream from the termination codon nor beyond a downstream initiation signal. RRF inactivation was bacteriostatic in the growing phase and bactericidal during the transition between the stationary and growing phase, confirming the essential nature of the fourth step of protein synthesis in vivo. [ABSTRACT FROM AUTHOR]

We describe the cloning and expression of a new cDNA from the filamentous fungus Aspergillus clavatus IFO 8605. This cDNA contains an open reading frame (ORF) that predicts a putative ribonuclease precursor with high similarity to the α-sarcin family of ribosome-inactivating proteins (RIPs). The cDNA encoding the mature protein was expressed in Escherichia coli, and the recombinant protein, a 17-kDa polypeptide designated clavin was purified and characterized. Clavin shows typical type-1 RIP properties: specific cleavage of ribosomal and synthetic RNA and inhibition of protein synthesis in cell-free and cellular systems. When selectively targeted to a tumour cell antigen by coupling to a monoclonal antibody (mAb) clavin was able to inhibit protein synthesis at nanomolar concentration. Pharmacokinetics analysis in mice indicated an elimination half-life (t1/2β) of 7.4 h with no particular accumulation in major organs. Liver toxicity was very limited and transient while no alteration of kidney function was observed. Clavin induced a late and very low antibody response in mice. The in vitro and in vivo biological characteristics of clavin, together with its availability in large amounts, suggest the usefulness of this toxin in the production of toxic chemical conjugates. [ABSTRACT FROM AUTHOR]

Ribosomal proteins L3 and L4 were purified to homogeneity from total protein isolated from the 50S subunit of Bacillus stearothermophilus by reversed-phase high-performance liquid chromatography (RP-HPLC). Amino acid sequences of both proteins were determined by automated N-terminal sequence analysis and sequencing of internal peptides. Using oligonucleotides deduced from the N-terminal region of protein L3 as hybridization probes, a DNA fragment coding for proteins L3, L4 and the N-terminal part of protein L23 has been identified, cloned and sequenced. The organization of the genes is identical to that found in the S10 operon of Escherichia coli. Comparison of the sequences of proteins L3 and L4 with those of other organisms revealed that all proteins of the L3 family are highly conserved. On the other hand, the archaebacterial L4 proteins show no significant sequence similarity to the E. coli L4 protein whereas the L4 protein of B. stearothermophilus is significantly similar to all of the L4 proteins and thus justifies the membership of all the L4 proteins in one protein family. The results are discussed with respect to the phylogenetic relationship between eubacteria, archaebacteria and eukaryotes and possible functional domains of proteins L3 and L4. [ABSTRACT FROM AUTHOR]

L2, L3, L4, L16 and L20 are proteins of the 50S ribosomal subunit of Escherichia coli which are essential for the assembly and activity of the peptidyl transferase centre. These proteins have been modified with the histidinespecific reagent, diethylpyrocarbonate, while L17 and LI8 were treated as controls. Each modified protein tested was able to participate in the reconstitution of a 50S particle when replacing its normal counterpart, although the particles assembled with modified L2 were heterogeneous. However, although they could support assembly, modified L16 and L20 were not themselves reconstituted stably, and modified L2 and L3 were found in less than stoichiometric amounts. Particles assembled in the presence of modified L16 retained significant peptidyl transferase activity (60-70% at 10 mM diethylpyrocarbonate) whereas those reconstituted with modified L2, L3, L4 or L20 had low activity (10-30% at 10 mM diethylpyrocarbonate). The particles assembled with the modified control portein, L17, retained 80% of their peptidyl transferase activity under the same conditions. The histidine residues within the essential proteins therefore contribute to ribosome structure and function in three significant ways; in the correct assembly of the ribosomal subunit (L2), for the stable assembly of the proteins within the ribosomal particle (L20 and L16 in particular), and directly or indirectly for the subsequent activity of the peptidyl transferase centre (L2, L3, L4 and L20). The essential nature of the unmodified histidines for assembly events precludes the use of the chemical-modification strategy to test the proposal that a histidine on one of the proteins might participate in the catalytic activity of the centre. [ABSTRACT FROM AUTHOR]

The subunit association capacity of 30S and 50S ribosomal subunits from Escherichia coli mutants lacking protein S20 or L11 as well as of 50S subunits depleted of L7/L12 was tested by sucrose gradient centrifugation and by a nitrocellulose filtration method based on the protection from hydrolysis with peptidyl-tRNA hydrolase of ribosome-bound AcPhe-tRNA. It was found that the subunits lacking either S20 or L11 display an altered association capacity, while the 50S subunits lacking L7/L12 have normal association behavior. The association of S20-lacking 30S subunits is quantitatively reduced, especially at low Mg2+ concentrations (5–12 mM), and produces loosely interacting particles which dissociate during sucrose gradient centrifugation. The association of L11-lacking 50S subunits is quantitatively near-normal at all Mg2+ concentrations and produces loosely associating particles only at low Mg2+ concentrations (5–8 mM); the mechanism of their association with 30S subunits, however, or the structure of the resulting 30S–50S couples is altered in such a way as to cause the ejection of an AcPhe-tRNA molecule pre-bound to the 30S subunits in response to poly(U). [ABSTRACT FROM AUTHOR]

Binding of the yeast Tyr-tRNA and Phe-tRNA to the A site, and the binding of their acetyl derivatives to the P site of poly(U11,A)-programmed Escherichia coli ribosomes was studied. Spermine stimulated the rate of binding of both tRNAs at least threefold, enabling more than 90% final saturation of both ribosomal binding sites. The effect is observed when the tRNAs, but not ribosomes or poly(U11,A), are preincubated with polyamine. Regardless of the binding site, optimal saturation was reached at spermine/tRNA molar ratios of 3 for tRNAPhe and 5 for tRNATyr. The same low spermine/tRNA ratios were previously reported to stabilize the conformation of these tRNAs in solution. On the other hand, the messenger-free, EF-Tu- and EF-G-dependent polymerization of lysine from E. coli Lys-tRNA is drastically reduced, while the poly(A)-directed polymerization is stimulated by spermine through a wide range of Mg2+ concentrations. Misreading of UUU codons as isoleucine, assayed by the A-site binding of E. coli Ile-tRNA, is also inhibited by spermine. All these results demonstrate that spermine increases the efficiency and accuracy of a series of macromolecular interactions leading to the correct incorporation of an amino acid into protein, at the same time preventing some unspecific or erroneous interactions. From the analogy with its known structural effects, it can be inferred that spermine does so by conferring on the tRNA a specific biologically functional conformation. [ABSTRACT FROM AUTHOR]

The locations of proteins BL1, BL6, BL23 and BL29 from Bacillus stearothermophilus have been determined on the ribosomal surface by immunoelectron microscopy. All four proteins were localized in the same region of the 50S subunit as their homologous counterparts from Escherichia coli, indicating that the ribosomal architecture is the same in both species. This finding is of great importance as it allows structural data obtained on ribosomes from either organism to be incorporated into a unique ribosome model. [ABSTRACT FROM AUTHOR]

The misincorporation of cysteine (codon: UGU/C) into twelve ribosomal proteins devoid of cysteine has been studied. Although it is generally assumed that cysteine is misincorporated at arginine and tryptophan residues (codons: CGU/U and UGG respectively), our results are consistent with the idea that cysteine is also mis-incorporated at phenylalanine residues (codon: UUU/C) through a second-position C : U mismatch. Cysteine was found in ribosomal proteins L29, L32/L33 and SW, under conditions where only its misincorporation at neutral residues was measured. Since these proteins contain no tryptophan, the data imply that cysteine has replaced a neutral amino acid other than tryptophan. Because there was a statistically significant correlation between the total level of cysteine in the twelve proteins under study and their content of phenylalanine and arginine residues, we conclude that there is a likelihood of cysteine misincorporation at phenylalanine residues, in addition to its misincorporation at arginine and tryptophan residues. Our measurements are consistent with the existence of a cluster of ribosomal proteins having an average mistranslation frequency of 2.5 × 10-4/residue and another having an average mistranslation frequency of 10-3 residue. There was three times less cysteine misincorporated into ribosomal protein LI than into L7/L12, although the LI mRNA contains eleven CGU/C codons and four UUU/C codons while the L7/L12 mRNA contains only one arginine and two phenylalanine codons (both proteins are free of tryptophan). Furthermore, the mRNAs for both LI and L7/L12 contain a CGU codon located in the context GUA-codon-GG and there was as much cysteine incorporated at this codon in L7/L12 [Bouadloun, F., Donner, D. and Kurland, C. G. (1983) EMBO J. 2, 1351 -1356] than in the whole of Li. This suggests that, relatively speaking, little cysteine is to be found at the phenylalanine and the other ten arginine positions of LI and that the phenylalanine residues of L7/L12 are particularly error-prone. [ABSTRACT FROM AUTHOR]

A and P sites of Escherichia coli ribosomes were titrated with AePhe-tRNAPhe, in the absence or presence of tetracycline. The P-site location of the bound AcPhe-tRNA was assessed by means of a quantitative puromycin reaction. The results demonstrate that, in agreement with the generally held view, tetracycline exclusively inhibits the A-site binding, if the statistical number of bound acyl-tRNA molecules per ribosome does not exceed about 0.5. However: above this value the P site becomes sensitive to tetracycline as well. It follows that the tightly coupled 70S ribosomes used in functional studies appear to be functionally heterogeneous, i.e. those P sites which cannot be affected by tetracycline are preferentially occupied by AcPhe-tRNA, whereas higher concentrations of this tRNA species are required to fill tetracycline-sensitive P sites. Furthermore, the results imply that under tRNA saturation conditions the tetracycline inhibition cannot be used as an indicator for the site location of bound tRNA. [ABSTRACT FROM AUTHOR]

The standard technique for determination of the ribosomal site location of bound tRNA, viz. the puromycin reaction, has been analyzed with regard to its applicability under tRNA saturation conditions. The criteria derived have been used to re-examine the exclusion principle for peptidyl-tRNA binding, which states that only one peptidyl-tRNA (AcPhe-tRNA) can be bound per ribosome although in principle two sites (A and P site) are available. The following results were obtained. 1. The puromycin reaction is only appropriate for a site determination if the reaction conditions prevent one ribosome from performing more than one puromycin reaction. With an excess of AcPhe-tRNA over ribosomes, and in the absence of EF-G, this criterion is fulfilled at 0 °C, where the P-site-bound material reacts with puromycin (quantitative reaction after 50 h), while the A-site-bound material does not. In contrast, at 37°C the extent of the puromycin reaction can exceed the binding values by 2&ndashh;4-fold (‘repetitive reaction’). In the presence of EF-G a repetitive puromycin reaction is seen even at 0 °C, i.e. EF-G can already promote a translocation reaction at 0 °C. However, the extent of translocation becomes negligibly low for short incubation times (up to 60 min) at 0 °C, if only catalytic amounts of EF-G are used. 2. Using the criteria outlined above, the validity of the exclusion principle for Escherichia coli ribosomes was confirmed pursuing two different experimental strategies. a) Ribosomes were saturated with AcPhe-tRNA at one molecule per 70S ribosome, and a quantitative puromycin reaction demonstrated the exclusive P-site location of the AcPhe-tRNA. The same result was also found in the presence of viomycin, which blocks the translocation reaction. These findings also indicate that here nearly 100% of the ribosomes participate in AcPhe-tRNA binding to the P site. b) Precharging the P sites of 70S ribosomes with one Ac[14C]Phe-tRNA molecule per ribosome prevented additional Ac[³H]Phe-tRNA binding. In contrast, 70S particles carrying one molecule of [14C]tRNAPhe per ribosome were able to bind up to a further 0.64 molecule Ac[³H]Phe-tRNA per ribosome. [ABSTRACT FROM AUTHOR]

Studies the efficiency and specificity of RNA-protein cross-linking in the 30S subunit of Escherichia coli ribosomes induced by low-intensity and high-intensity photons. Observations of biphotonic processes in the high-intensity photon environment; Efficiency of cross-linking is 20-100 higher upon two-step excitation compared with single-step excitation.

Small ribosomal subunits of gram-positive cells of Streptomyces aureofaciens contain an acidic protein designated SS1. Purified protein SS1 has the same mobility in sodium dodecyl sulfate/polyacrylamide gel as ribosomal protein SI of Escherichia coli (apparent Mr, 68000). Protein SS1 was dissected under mild conditions with trypsin and generated fragments were compared with well-characterized fragments of protein S1. The protein SS1 contains a structure homologous with the C-terminal fragment of protein SI. The affinity of protein SS1 to poly(U) is virtually identical with that of E. eoli protein SI. In contrast to protein S1, the addition of SS1 to partially S1-depleted ribosomes of E. coli had no stimulatory effect on poly(U)- directed synthesis of polyphenylalanine. At molar excess of SS1 over ribosomes, the protein had comparable inhibitory effect on polypeptide synthesis as had SI of E. coli. Ribosomes of S. aureofaciens required about one order of magnitude higher concentration of poly(U) for maximum synthetic activity than did ribosomes of E. coli. The addition of proteins SS1 or S1 to ribosomes of S. aureofaciens had no stimulatory effect on translation of poly(U). Our data indicate that the high-molecular-mass acidic protein SS1 of small ribosomal subunits of S. aureofaeiens exhibits only a part of the functional properties of E. coli protein S1. [ABSTRACT FROM AUTHOR]

Photoactivation of the [³H]dihydrorosaramicin chromophore at a wavelength above 300 nm allows the covalent attachment of the macrolide antibiotic to the bacterial ribosome. Bidimensional electrophoresis shows that the radioactivity is mainly associated with proteins L1, L5, L6, L15, L18, L19, S1, S3, S5, S5 and S9. When photoincorporation of the drug is conducted in the presence of puromycin as effector of [³h]dihydrorosaramicin-binding sites, a decrease in the labeling of most proteins is observed, except for L18 and L19, which are radiolabeled to a larger extent. These results allow us to speculate that L18 and L19 belong to the high-affinity binding site of rosaramicin antibiotic. [ABSTRACT FROM AUTHOR]

The mutant strain of Escherichia coli, TP28, synthesises ribosomes by an abnormal pathway and accumulates large quantities of 47S ribonucleoprotein particles. The protein complement of mutant 70S ribosomes is normal but 47S particles contain only traces of proteins L28 and L33 and have a significantly reduced content of four other proteins. The mutation reduces the rates of synthesis of L28 and L33 by about half but other widespread alterations ensue. In particular, ribosomal protein synthesis in the mutant strain becomes less well balanced than in its parent: some proteins, particularly those from promoter-proximal genes, are oversynthesized and their excess then degraded. [ABSTRACT FROM AUTHOR]

The ribosomal environment of the N-terminus of the nascent polypeptide chain has been investigated using peptides of different lengths, synthesized in situ on Escherichia coli ribosomes; the peptides each carry a photoreactive diazirine moiety at their N-terminus, so as to generate cross-links to neighbouring ribosomal components. Our previous studies [Choi, K. M. & Brimacombe, R. (1998) Nucleic Acids Res. 26, 887-895] with three independent families of peptides, derived from the E. coli ompA protein gene, the tetracycline-resistance gene and the bacteriophage T4 gene 60, identified a series of sites within the 23S rRNA to which the peptides became cross-linked. The distribution of these cross-links indicated that the nascent peptide is very flexible within the 50S subunit. Here, we demonstrate that the N-termini of the ompA and gene-60 peptides can, in addition, even become concomitantly cross-linked to the 30S subunit. The cross-linking is predominantly to 30S ribosomal proteins S1, S2, S4 and (to a lesser extent) S3, which form a cluster near to the decoding region. This result is discussed in terms of the flexibility of the nascent peptide during the co-translational folding process, and in terms of the ’ribosomal bypass' phenomenon which is known to occur during translation of the gene 60 mRNA. [ABSTRACT FROM AUTHOR]

A 6-kb DNA fragment from an extreme thermophile, Thermus thermophilus, carrying the genes for cytochrome oxidase ba3 subunit I (cbaA) and the ribosomal protein S15 (rpsO) was cloned into Escherichia coli. The gene rpsO was sequenced. The deduced amino acid sequence exhibits 59% identity to the corresponding protein from E. coli. Expression of rpsO in E. coli requires the use of a fully repressed inducible promoter because S15 from T. thermophilus is toxic for E. coli cells, When purified without denaturation from either overproducing E. coli strain or from T. thermophilus ribosomes, the S15 protein is stable and binds a cloned T. thermophilus 16S rRNA fragment (nucleotides 559-753), with low identical dissociation constants (2.5 nM), thus demonstrating that the thermophilic protein folds correctly in a mesophilic bacterium. The rRNA fragment bound corresponds in position and structure to the 16S rRNA fragment of E. coli. A similar high affinity was also found for the binding of S 15 from T. thermophilus or E. coli to the corresponding E. coli 16S rRNA fragment, whereas a slightly lower affinity was observed in binding experiments between E. coli S15 and T. thermophilus 16S rRNA fragment. These results suggest that S15 from T. thermophilus recognizes similar determinants in both rRNA fragments. Competition experiments support this conclusion. [ABSTRACT FROM AUTHOR]

The Escherichia coli cspA gene, encoding the major cold-shock protein CspA, was deprived of its natural promoter and placed in an expression vector under the control of the inducible λ PL promoter. After induction of transcription by thermal inactivation of the λ ts repressor, abundant expression of the product (CspA) was obtained if the cells were subsequently incubated at 10°C, but poor expression was obtained if the cells were incubated at 37°C or 30°C. The reason for this differential temperature-dependent expression was investigated and it was found that: (i) the CspA content of the cells decreased more rapidly at 37°C compared to 10°C, regardless of whether transcription was turned off by addition of rifampicin; (ii) both the chemical and functional half-lives of the cspA transcript were substantially longer at 10°C compared to 37°C; (iii) S30 extracts as well as 70S ribosomes prepared from cold-shocked cells translated CspA mRNA (but not phage MS2 RNA) more efficiently than equivalent extracts or ribosomes obtained from control cells grown at 37°C; and (iv) purified CspA stimulated CspA mRNA translation. Overall, these results indicate that a selective modification of the cold-shocked translational apparatus favouring translation of CspA mRNA, and an increased stability of this mRNA at low temperature, may play an important role in the induction of cspA expression during cold shock. [ABSTRACT FROM AUTHOR]